Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of cGMP-dependent protein kinase (PKG) on recombinant human alpha 1 beta 2 gamma 2L GABAA receptors expressed in Xenopus oocytes was studied using the two-electrode voltage-clamp technique. The cGMP analog 8BrcGMP (1 mM) produced an increase in GABA-gated chloride currents. Intracellular injection of the PKG inhibitor peptide, PKGI, prevented the 8BrcGMP-mediated increase in the GABA response indicating that 8BrcGMP enhances GABAA receptor function via activation of PKG. Previous studies have shown that PKG phosphorylates a fusion protein corresponding to the intracellular loop of the beta 1 subunit [McDonald and Moss, J. Biol. Chem., 269 (1994) 18111-18117]. In the present study, site-directed mutagenesis of this phosphorylation site (beta 2ser410) failed to eliminate the effects of 8BrcGMP on the GABA response. These results suggest that there may be other sites on the receptor which are regulated by PKG or that PKG phosphorylates other proteins which may influence GABAA receptor function.
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PMID:Effect of PKG activation on recombinant GABAA receptors. 891 90

1. Protein kinase modulation of gamma-aminobutyric acid-A (GABAA)- and glycine-activated Cl- currents in freshly dissociated, morphologically identified rabbit retinal rod bipolar cells was studied under voltage clamp with the use of the whole cell patch-clamp technique. Responses to pulses of GABA and glycine were monitored before, during, and after application of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC) activators, inactive analogues, and inhibitors. 2. Bath perfusion with either forskolin, an adenylate cyclase activator, or its inactive analogue, 1,9 dideoxyforskolin, reduced the GABA-activated Cl- currents by 30-50%; coapplication of N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8), a PKA inhibitor, did not prevent the forskolin effects. The membrane-permeable cAMP analogues, 8-bromo-cAMP and 8-(4-Chlorophenylthio)-cAMP, and intracellularly dialyzed cAMP, did not modulate either the GABA- or glycine-activated Cl- current. Perfusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) had no direct effect on the GABA-activated current and did not alter the results with cAMP or its membrane-permeable analogues. Collectively, these results make it very unlikely that PKA represents an important mechanism of either GABAA or glycine channel modulation in the rabbit rod bipolar cell. 3. Although the isoquinoline sulfonamide protein kinase inhibitor H-8 was without discernible effect, the related compounds 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochlorine (H-7) and N-(2-Aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9) both dramatically reduced the GABA response. H-7 also strongly reduced the response to glycine, whereas H-8 had no effect and H-9 had an intermediate effect. Because only certain members of this inhibitor class of agents proved effective, and their effectiveness appeared unrelated to the established activity profiles, these agents probably inhibit the Cl- currents in a phosphorylation-independent manner. Direct interaction of these inhibitors with binding sites in the GABAA receptor-channel complex has been previously reported in some other preparations. 4. The phorbol ester and PKC activator phorbol 12,13 dibutyrate (PDB) led to a 35-55% reduction in the GABA-activated Cl- current of the rod bipolar cell. The broad-spectrum kinase inhibitor staurosporine, and the more PKC-specific inhibitor calphostin C, had no direct effect on GABA responses, but prevented Cl- current reduction when coapplied with PDB. Phorbol 12-myristate 13-acetate (PMA) reduced the GABA-activated current in a fashion very similar to PDB. Staurosporine and calphostin C blocked the PMA effect. No reduction of Cl- current was seen with the inactive analogue, 4-alpha-PMA, used as a control for PKC-independent phorbol ester effects. 5. PDB effectively reduced the GABA-activated Cl- current of the rod bipolar cell at low concentrations, whereas PMA had a diminished effect at low concentrations. This is consistent with the reported concentration-dependent abilities of these agents to promote translocation of PKC-alpha immunoreactivity from the membrane to the cytosolic compartment in the rabbit retinal rod bipolar cell. Collectively, the data from phorbol esters, inactive analogues, and kinase inhibitors support the existence of a PKC-mediated mechanism for GABA-activated Cl- current reduction in these cells. 6. The naphthalenesulfonamide PKC activator N-(n-Heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) also potently and reversibly reduced the GABA-activated current. Staurosporine and calphostin C eliminated this effect. When the nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTP-gamma-S) replaced GTP in the recording pipette, the SC-10-mediated GABA current reduction became irreversible.(ABSTRACT TRUNCATED)
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PMID:Protein kinase modulation of GABAA currents in rabbit retinal rod bipolar cells. 893 Feb 56

Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7-10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5-10 microM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25-0.5 mM). GABA (10 microM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 microM) and vapreotide (RC 160; 10 microM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 microM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 microM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5-10 microM). Finally, both specific (Rp-8-bromoguanosine 3',5'-monophosphate, 10 microM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 microM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca2+ level.
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PMID:Neuroprotective effect of somatostatin on nonapoptotic NMDA-induced neuronal death: role of cyclic GMP. 897 41

Opioid receptors located on interneurons in the ventral tegmental area (VTA) inhibit GABA(A)-mediated synaptic transmission to dopamine projection neurons. The resulting disinhibition of dopamine cells in the VTA is thought to play a pivotal role in drug abuse; however, little is known about how this GABAA synapse is affected after chronic morphine treatment. The regulation of GABA release during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. Slices containing the VTA were prepared and maintained in morphine-free solutions, and GABAA IPSCs were recorded from dopamine cells. The amplitude of evoked IPSCs and the frequency of spontaneous miniature IPSCs measured in slices from morphine-treated guinea pigs were greater than placebo-treated controls. In addition, activation of adenylyl cyclase, with forskolin, and cAMP-dependent protein kinase, with Sp-cAMPS, caused a larger increase in IPSCs in slices from morphine-treated animals. Conversely, the kinase inhibitors staurosporine and Rp-CPT-cAMPS decreased GABA IPSCs to a greater extent after drug treatment. The results indicate that the probability of GABA release was increased during withdrawal from chronic morphine treatment and that this effect resulted from an upregulation of the cAMP-dependent cascade. Increased transmitter release from opioid-sensitive synapses during acute withdrawal may be one adaptive mechanism that results from prolonged morphine treatment.
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PMID:Increased probability of GABA release during withdrawal from morphine. 898 1

The effects of nefiracetam on GABA-induced chloride currents were studied with rat dorsal root ganglion neurons in primary culture using the whole-cell patch-clamp technique. The dose-response curve for GABA-induced currents was shifted by 16 microM to lower concentrations by 10 microM nefiracetam while the maximal response was reduced by 22.84 +/- 0.68%. Thus at a low concentration (10 microM) of GABA, the chloride currents were potentiated by nefiracetam in a concentration-dependent manner. With 10 microM nefiracetam, the potentiation occurred slowly and the recovery after washout was also slow. The desensitization of the GABAA receptor at high concentration (100 microM) of GABA was accelerated by nefiracetam. The recovery process of chloride currents from desensitization was not affected by nefiracetam. KT 5720 (0.56 microm), a specific protein kinase A (PKA) inhibitor, blocked the transient potentiation of GABA-activated currents by nefiracetam, but did not affect the acceleration of desensitization. Nefiracetam suppression of GABA-induced currents was also abolished by KT 5720 or the pertussis toxin. Thus, nefiracetam may inhibit Gi/G(o) proteins leading to a cascade of events that increase the intracellular cAMP level, activate the PKA system, and suppress GABA-induced currents. Nefiracetam-induced transient potentiation and acceleration of desensitization of GABA-induced currents may involve other pathways. The nefiracetam modulation of the GABAA receptor function will result in a nootropic effect on the central nervous system through modification of synaptic transmission.
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PMID:Effects of the nootropic drug nefiracetam on the GABAA receptor-channel complex in dorsal root ganglion neurons. 901 40

The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity.
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PMID:A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells. 903 53

The effect of baclofen on the function of the gamma-aminobutyric acidA (GABAA) receptor was examined in acutely dissociated neurons of bullfrog dorsal root ganaglia (DRG) by using the whole-cell voltage-clamp method. Baclofen (0.1-100 microM) depressed the inward currents produced by GABA (100 microM) and muscimol (100 microM). Baclofen shifted the concentration-response curve for GABA (1 microM-1 mM) downward. Baclofen decreased the maximum response (Vmax) to GABA without changing the apparent dissociation constant (Kd), suggesting a noncompetitive antagonism. The effect of baclofen on the GABA current was blocked by antagonists for the GABAB receptor; the rank order of potency was P-[3-Aminopropyl]-P-diethoxymethylphosphinic acid (CGP 55845A) > > 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P- benzyl-phosphinic acid (CGP 35348) > saclofen > > phaclofen. Baclofen produced an irreversible depression of the GABA current in neurons dialyzed with an internal solution containing guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 100 microM). Intracellular guanosine 5'-O-(2-thiodiphosphate) (GDP beta S, 100 microM) blocked the inhibitory effect of baclofen on the GABA current. Forskolin (10 microM) and dibutyryl N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophophate (db-cyclic AMP) (200 microM) depressed the GABA current. N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9, 40 microM) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 50 microM), protein kinase A (PKA) inhibitors, reduced the depressant effect of baclofen on the GABA current. The baclofen-induced depression of the GABA current was blocked by PKI(5-24), a specific PKA inhibitor, but not by PKC(19-36), a specific protein kinase C (PKC) inhibitor. We suggest that GABAB receptors regulate the GABAA receptor function through a G-protein linked to the adenylyl cyclase-PKA pathway in bullfrog DRG neurons.
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PMID:Baclofen reduces GABAA receptor responses in acutely dissociated neurons of bullfrog dorsal root ganglia. 913 75

In the majority of developing neurons, GABA can exert depolarizing actions, thereby raising neuronal Ca2+. Ca2+ elevations can have broad consequences during development, inducing gene expression, altering neurite outgrowth and growth cone turning, activating enzyme pathways, and influencing neuronal survival. We used fura-2 and fluo-3 Ca2+ digital imaging to assess the effects of inhibiting or activating the cAMP signal transduction pathway on GABA activity mediating Ca2+ rises during the early stages of in vitro hypothalamic neural development. Our experiments stemmed from the finding that stimulation of transmitter receptors shown to either activate or inhibit adenylyl cyclase activity caused a rapid decrease in Ca2+ rises mediated by synaptically released GABA. Both the adenylyl cyclase activator forskolin and the inhibitor SQ-22,536 reduced the Ca2+ rise elicited by the synaptic release of GABA. Bath application of the membrane-permeable cAMP analogs 8-bromo-cAMP (8-Br-cAMP) or 8-(4-chlorophenylthio)-cAMP (0.2-5 mM) produced a rapid, reversible, dose-dependent inhibition of Ca2+ rises triggered by synaptic GABA release. Potentiation of GABAergic activity mediating Ca2+ rises was observed in some neurons at relatively low concentrations of the membrane-permeable cAMP analogs (20-50 microM). In the presence of tetrodotoxin (TTX), postsynaptic Ca2+ rises triggered by the bath application of GABA were only moderately depressed (13%) by 8-Br-cAMP (1 mM), suggesting that the inhibitory effects of 8-Br-cAMP were largely the result of a presynaptic mechanism. The protein kinase A (PKA) inhibitors H89 and Rp-3', 5'-cyclic monophosphothioate triethylamine also caused a large reduction (>70%) in Ca2+ rises triggered by synaptic GABA release. Unlike the short-term depression elicited by activation of the cAMP signal transduction pathway, Ca2+ depression elicited by PKA inhibition persisted for an extended period (>30 min) after PKA inhibitor washout. Postsynaptic depression of GABA-evoked Ca2+ rises triggered by H89 (in the presence of TTX) recovered rapidly, suggesting that the extended depression observed during synaptic GABA release was largely through a presynaptic mechanism. Long-term Ca2+ modulation by cAMP-regulating hypothalamic peptides may be mediated through a parallel mechanism. Together, these results suggest that GABAergic activity mediating Ca2+ rises is dependent on ongoing PKA activity that is maintained within a narrow zone for GABA to elicit a maximal Ca2+ elevation. Thus, neuromodulator-mediated changes in the cAMP-dependent signal transduction pathway (activation or inhibition) could lead to a substantial decrease in GABA-mediated Ca2+ rises during early development.
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PMID:GABA activity mediating cytosolic Ca2+ rises in developing neurons is modulated by cAMP-dependent signal transduction. 916 37

1. The modulation by adenosine of GABA-activated current (IGADA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. 2. In most of the DRG neurons examined (68/90, 75.5%) adenosine (1-10 microM) suppressed IGABA, while in some neurons examined, it potentiated (16/90, 17.8%) IGABA. It exerted no effects on IGABA in a few cells (6/90, 6.7%). 3. Adenosine shifted the GABA concentration-response curve downward with no significant change of the EC50. The maximal response to GABA was suppressed by 29.6 +/- 2.6%. The adenosine-induced inhibition of IGABA showed no voltage dependence. 4. 8-Cyclopentyl-1,3-dimethylxanthine (DPCPX; 1 microM), a selective A1 adenosine receptor antagonist, partially reversed adenosine inhibition of IGABA and completely blocked N6-cyclo-hexyladenosine (CHA; an A1 adenosine receptor agonist) inhibition of IGABA. DPCPX (1 microM) also blocked the suppression of IGABA by 2-chloroadenosine (CADO). CGS21680, a selective A2A adenosine receptor agonist, did not inhibit IGABA and DMPX, a selective A2A adenosine receptor antagonist, did not prevent adenosine inhibition of IGABA. 5. Intracellular application of H-7 (20 microM; a protein kinase C inhibitor) reversed adenosine inhibition of IGABA while inclusion of cAMP (1 mM), H-9 (20 microM; a protein kinase A inhibitor) and BAPTA (10 mM; a chelator of calcium ions) in the recording pipette did not affect the depression of IGABA by adenosine. IGABA was also suppressed by internal perfusion of PMA, a protein kinase C activator. 6. The results suggest that adenosine, as a neuromodulator, exerts a modulatory effect on the GABA-induced presynaptic inhibition in primary sensory transmission.
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PMID:Modulation by adenosine of GABA-activated current in rat dorsal root ganglion neurons. 917 95

1. The A type of acetylcholine response of Helix neurons is downmodulated by low concentrations of GABA that do not elicit any measurable change in membrane potential or conductance. 2. We find that these physiological actions are associated with an increase in both intracellular cyclic AMP levels and 45Ca2+ influx. 3. The modulation of the acetylcholine response by GABA is blocked when the neurons are injected with EGTA to prevent a rise in intracellular Ca2+ concentration or when tolbutamide, an inhibitor of protein kinase A, is applied. 4. These results are consistent with the effects of GABA being mediated by a metabotropic GABA receptor that is activated at very low GABA concentrations and mediates modulation of the acetylcholine response via regulation of intracellular Ca2+ and cyclic AMP levels.
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PMID:Metabotropic GABA receptors regulate acetylcholine responses on snail neurons. 919 95


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